Macular degeneration diagnostics and therapeutics

ABSTRACT

Therapeutics and diagnostics based on the identification of genetic mutations, which cause Macular Degeneration (MD) are disclosed.

1. BACKGROUND OF THE INVENTION

Macular degeneration is a clinical term that is used to describe avariety of diseases that are all characterized by a progressive loss ofcentral vision associated with abnormalities of Bruch's membrane and theretinal pigment epithelium. These disorders include very commonconditions that affect older patients (age related macular degenerationor AMD) as well as rarer, earlier-onset dystrophies that in some casescan be detected in the first decade of life¹⁻¹⁸. The genes associatedwith some of these dystrophies have been mapped,⁵⁻¹⁴ and in four cases,blue-cone monochromasy,¹⁵ pattern dystrophy,^(16,17) and Sorsby fundusdystrophy,¹⁸ and Best Disease actually identified. However, none of thelatter genes has been found to be responsible for a significant fractionof typical late-onset macular degeneration.

In developed countries, AMD is the most common cause of legal blindnessin older patients.¹⁹ The hallmark of this condition is the presence ofdrusen, which are ophthalmoscopically visible, yellow-white hyalineexcrescences of Bruch's membrane. In some families, drusen are heritablein an autosomal dominant fashion.

In 1875, Hutchinson and Tay published a paper entitled “SymmetricalCentral Choroido-Retinal Disease Occurring in Senile Persons”.²⁰ Thispaper includes one of the first descriptions of the constellation ofclinical findings: now known as age related macular degeneration (AMD).Specifically, three of the ten patients in the report were sistersaffected with whitish spots (now referred to as drusen) in the macula.In 1899, Doyne²¹ a similar disorder in which the abnormal spots werenearly confluent such that the macula had a, “honeycomb” appearance.Histopathologic examination of one of Doyne's patients²² revealed theabnormalities to be hyaline thickenings of Bruch's membrane. In 1925,Vogt²³ published the first description of the ophthalmoscopic appearanceof a form of familial drusen that had been observed in patients livingin the Leventine valley in the Ticino canton of southern Switzerland.Klainguti²⁴ fully characterized this condition in 1932 and demonstratedits autosomal dominant inheritance. This disorder eventually becameknown as malattia leventinese, “ML” (i.e., Leventine disease). 1948,Waardenburg²⁵ stated that there was little reason to make a distinctionbetween malattia leventinese and the condition described by Doyne,referred to as Doyne's Honeycomb Retinal Dystrophy (DHRD). This positionwas strengthened when Forni and Babel²⁶ found that the histopathologicfeatures of malattia leventinese were indistinguishable from those ofDoyne's honeycomb choroiditis. Piguet, Haimovici and Bird²⁷ recentlyreviewed the history of these conditions and also pointed out that thedrusen in families with malattia leventinese are frequently distributedin a radical pattern. Choroidal neovascularization is uncommon inpatients with radial drusen but does occur.²⁷ Although originallyrecognized in Switzerland, families affected with autosomal dominantradial drusen have been identified in Czechoslovakia,^(28,29) and theUnited States.³⁰

In 1996, ML was mapped to chromosome 2p16-21⁴⁸. Shortly thereafter, DHRDwas mapped to the same: locus⁴⁹ and the genetic interval was narrowed^(48,49). ML and DHRD are very similar phenotypically to AMD.

Currently, there is no therapy that is capable of significantly slowingthe degenerative progression of macular degeneration, and treatment islimited to laser photocoagulation of the subretinal neovascularmembranes that occur in 10-15% of affected patients.

2. SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of anovel human gene encoding a novel human protein, which has sequencehomologies with fibulin (1 and 2), fibrillin, nidogen, notch, protein Sand Factor IX, The newly identified proteins and nucleic acids describedherein are referred to as “EFEMPs”. The human EFEMP1 gene (hereinreferred to as hEFEMP1) transcript is shown in FIG. 5 and includes 5′and 3′ untranslated regions and a 1479 base pair open reading frameencoding a 493 amino acid polypeptide having SEQ ID NO. 1. Mouse EFEMP1is expressed in eye, brain, heart, lung and kidney tissue. A nucleicacid comprising the cDNA encoding the full length human EFEMP1polypeptide was deposited at the American Type Culture Collection on______ and has been assigned ATCC Designation No. ______.

In one aspect, the invention features isolated EFEMP1nucleic acidmolecules. In one embodiment, the EFEMP1 nucleic acid is from avertebrate. In a preferred embodiment, the EFEMP1 nucleic acid is from amammal, e.g. a human. In an even more preferred embodiment, the nucleicacid has the nucleic acid sequence set forth in FIG. 5 or a portionthereof. The disclosed molecules can be non-coding, (e.g. a probe,antisense, or ribozyme molecule) or can encode a functional EFEMP1polypeptide (e.g. a polypeptide which specifically modulates biologicalactivity, by acting as either agonist or antagonist of at least onebioactivity of the human EFEMP1 polypeptide). In one embodiment thenucleic acid molecule can hybridize to the EFEMP1 gene contained in ATCCdesignation number ______. In another embodiment, the nucleic acid ofthe present invention can hybridize to a vertebrate EFEMP1 gene or tothe complement of a vertebrate EFEMP1 gene. In a further embodiment, theclaimed nucleic acid can hybridize with a nucleic acid sequence shown inFIG. 5 or a complement thereof. In a preferred embodiment, thehybridization is conducted under mildly stringent or stringentconditions.

In further embodiments, the nucleic acid molecule is an EFEMP1 nucleicacid that is at least about 70%, preferably about 80%, more preferablyabout 85%, and even more preferably at least about 90% or 95% homologousto the nucleic acid shown as FIG. 5 or to the complement of the nucleicacid shown as FIG. 5. In a further embodiment, the nucleic acid moleculeis an EFEMP1 nucleic acid that is at least about 70%, preferably atleast about 80%, more preferably at least about 85% and even morepreferably at least about 90% or 95% similar in sequence to the EFEMP1nucleic acid contained in ATCC designation number ______.

The invention also provides probes and primers comprising substantiallypurified. oligonucleotides, which correspond to a region of nucleotidesequence which hybridizes to at least about 6, at least about 10, atleast about 15, at least about 20, or preferably at least about 25consecutive nucleotides of the sequence set forth as FIG. 5 orcomplements of the sequence set forth as FIG. 5 or naturally occurringmutants or allelic variants thereof. In preferred embodiments, theprobe/primer further includes a label group attached thereto, which iscapable of being detected.

For expression, the subject nucleic acids can be operably linked to atranscriptional regulatory sequence, e.g., at least one of atranscriptional promoter (e.g., for constitutive expression or inducibleexpression) or transcriptional enhancer sequence. Such regulatorysequences in conjunction with an EFEMP1 nucleic acid molecule canprovide a useful vector for gene expression. This invention alsodescribes host cells transfected with said expression vector whetherprokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo(e.g transgenic) methods for producing EFEMP1 proteins by employing saidexpression vectors.

In another aspect, the invention features isolated EFEMP1 polypeptides,preferably substantially pure preparations, e.g. of plasma purified orrecombirantly produced polypeptides. The EFEMP1 polypeptide can comprisea full length protein or can comprise smaller fragents corresponding toone or more particular motifs/domains, or fragments comprising at leastabout 6, 10, 25, 50, 75, 100, 125, 150, 200, 225, 250, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480 or 490 amino acids in length. In particularly preferredembodiments, the subject polypeptide has an EFEMP1 bioactivity.

In a preferred embodiment, the polypeptide is encoded by a nucleic acidwhich hybridizes with the nucleic acid sequence represented in FIG. 5.In a further preferred embodiment, the EFEMP1 polypeptide is comprisedof the amino acid sequence set forth in SEQ ID NO. 1. The subject EFEMP1protein also includes within its scope modified proteins, e.g. proteinswhich are resistant to post-translational modification, for example, dueto mutations which alter modification sites (such as tyrosine,threonine, serine or aspargine residues), or which prevent glycosylationof the protein, or which prevent interaction of the protein withintracellular proteins involved in signal transduction.

The EFEMP1 polypeptides of the present invention can be glycosylated, orconversely, by choice of the expression system or by modification of theprotein sequence to preclude glycosylation, reduced-carbohydrate analogscan also-be provided. Glycosylated forms can be obtained based onderivatization with glycosaminoglycan chains. Also, EFEMP1 polypeptidescan be generated which lack an endogenous signal sequence (though thisis typically cleaved off even if present in the pro-form of theprotein).

In yet another preferred embodiment, the invention features a purifiedor recombinant polypeptide, which has the ability to modulate, e.g.,mimic or antagonize, an activity of a wild-type EFEMP1 protein.Preferably, the polypeptide comprises an amino acid sequence identicalor homologous to a sequence designated in SEQ ID NO. 1.

Another aspect of the invention features chimeric molecules (e.g.,fusion proteins) comprising an EFEMP1 protein. For instance, the EFEMP1protein can be provided as a recombinant fusion protein which includes asecond polypeptide portion, e.g., a second polypeptide having an aminoacid sequence unrelated (heterologous) to the EFEMP1 polypeptide. Apreferred EFEMP1 fusion protein is an immunoglobulin-EFEMP1 fusionprotein, in which an immunoglobulin constant region is fused to anEFEMP1 polypeptide.

Yet another aspect of the present invention concerns an immunogencomprising an EFEMP1 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for anEFEMP1 polypeptide; e.g. a humoral response, an antibody response and/orcellular response. In a preferred embodiment, the immunogen comprises anantigenic determinant, e.g. a unique determinant of a protein encoded bythe nucleic acid set forth in FIG. 5 or as set forth in SEQ ID NO. 1.

A still further aspect of the present invention features antibodies andantibody preparations specifically reactive with an eipitope of anEFEMP1 protein.

The invention also features transgenic non-human animals which include(and preferably express) a heterologous form of an EFEMP1 gene describedherein, or which misexpress an endogenous EFEMP1 gene (e.g., an animalin which expression of one or more of the subject MFGF proteins isdisrupted). Such transgenic animals can serve as animal models forstudying cellular and/or tissue disorders comprising mutated ormis-expressed EFEMP1 alleles or for use in drug screening.Alternatively, such transgenic animals can be useful for expressingrecombinant EFEMP1 polypeptides.

The invention further features assays and kits for determining whetheran individual's EFEMP1 genes and/or proteins, are defective or deficient(e.g in activity and/or level), and/or for determining the identity ofEFEMP1 alleles. In one embodiment, the method comprises the step ofdetermining the level of EFEMP1 protein, the level of EFEMP1 mRNA and/orthe transcription rate of an EFEMP1 gene. In another preferredembodiment, the method comprises detecting, in a tissue of the subject,the presence or absence of a genetic alteration, which is characterizedby at least one of the following: a deletion of one or more nucleotidesfrom a gene; an addition of one or more nucleotides to the gene; asubstitution of one or more nucleotides of the gene; a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; and/or a non-wild type levelof the EFEMP1 protein. For example, detecting a genetic alteration orthe presence of a specific polymorphic region can include (i) providinga probe/primer comprised of an oligonucleotide which hybridizes to asense or antisense sequence of an EFEMP1 gene or naturally occurringmutants thereof, or 5′ or 3′ flanking sequences naturally associatedwith the EFEMP1 gene; (ii) contacting the probe/primer with anappropriate nucleic acid containing sample; and (iii) detecting, byhybridization of the probe/primer to the nucleic acid, the presence orabsence of the genetic alteration. Particularly preferred embodimentscomprise: 1) sequencing at least a portion of an EFEMP1 gene, 2)performing a single strand conformation polymorphism (SSCP) analysis todetect differences in electrophoretic mobility between mutant andwild-type nucleic acids; and 3) detecting or quantitating the level ofan EFEMP1 protein in an immunoassay using an antibody which isspecifically immunoreactive with a wild-type or mutated EFEMP1 protein.

Information obtained using the diagnostic assays described herein (aloneor in conjunction with information on another genetic defect, whichcontributes to the same disease) is useful for diagnosing or confirmingthat a symptomatic subject has a genetic defect (e.g. in an EFEMP1 geneor in a gene that regulates the expression of an EFEMP1 gene), whichcauses or contributes to the particular disease or disorder.Alternatively, the information (alone or in conjunction with informationon another genetic defect, which contributes to the same disease) can beused prognostically for predicting whether a non-symptomatic subject islikely to develop a disease or condition, which is caused by orcontributed to by an abnormal EFEMP1 activity or protein level in asubject (e.g. a macular degeneration). In particular, the assays permitone to ascertain an individual's predilection to develop a conditionassociated with a mutation in EFEMP1, where the mutation is a singlenucleotide polymorphism (SNP). Based on the prognostic information, adoctor can recommend a regimen (e.g. diet or exercise) or therapeuticprotocol usefull for preventing or prolonging onset of the particulardisease or condition in the individual.

In addition, knowledge of the particular alteration or alterations,resulting in defective or deficient EFEMP1 genes or proteins in anindividual, alone or in conjunction with information on other geneticdefects contributing to the same disease (the genetic profile of theparticular disease) allows customization of therapy for a particulardisease to the individual's genetic profile, the goal ofpharmacogenomics. For example, an individual's EFEMP1 genetic profile orthe genetic profile of a disease or condition to which EFEMP1 geneticalterations cause or contribute, can enable a doctor to: 1) moreeffectively prescribe a drug that will address the molecular basis ofthe disease or condition; and 2) better determine the appropriate dosageof a particular drug. For example, the expression level of EFEMP1proteins, alone or in conjunction with the expression level of othergenes known to contribute to the same disease, can be measured in manypatients at various stages of the disease to generate a transcriptionalor expression profile of the disease. Expression patterns of individualpatients can then be compared to the expression profile of the diseaseto determine the appropriate drug and dose to administer to the patient.

The ability to target populations expected to show the highest clinicalbenefit, based on the EFEMP1 or disease genetic profile, can enable: 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of EFEMP1 as a marker is useful for optimizing effectivedose).

In another aspect, the invention provides methods for identifying acompound which modulates an EFEMP1 activity, e.g. the interactionbetween an EFEMP1 polypeptide and a target peptide In a preferredembodiment, the method includes the steps of (a) forming a reactionmixture including: (i) an EFEMP1 polypieptide, (ii) an EFEMP1 bindingpartner, and (iii) a test compound; and (b) detecting interaction of theEFEMP1 polypeptide and the EFEMP1 binding protein. A statisticallysignificant change (potentiation or inhibition) in the interaction ofthe EFEMP1 polypeptide and EFEMP1 binding protein in the presence of thetest compound, relative to the interaction in the absence of the testcompound, indicates a potential agonist (mimetic or potentiator) orantagonist (inhibitor) of EFEMP1 bioactivity for the test compound. Thereaction mixture can be a cell-free protein preparation, e.g., areconstituted protein mixture or a cell lysate, or it can be arecombinant cell including a heterologous nucleic acid recombinantlyexpressing the EFEMP1 binding partner.

In preferred embodiments, the step of detecting interaction of theEFEMP1 and EFEMP1 binding partner is a competitive binding assay. Inother preferred embodiments, at least one of the EFEMP1 polypeptide andthe EFEMP1 binding partner comprises a detectable label, and interactionof the EFEMP1 and EFEMP1 binding partner is quantified by detecting thelabel in the complex. The detectable label can be, e.g., a radioisotope,a fluorescent compound, an enzyme, or an enzyme co-factor. In otherembodiments, the complex is detected by an immunoassay.

Yet another exemplary embodiment provides an assay for screening testcompounds to identify agents which modulate the amount of EFEMP1produced by a cell. In one embodiment, the screening assay comprisescontacting a cell transfected with a reporter gene operably linked to anEFEMP1 promoter with a test compound and determining the level ofexpression of the reporter gene. The reporter gene can encode, e.g., agene product that gives rise to a detectable signal such as: color,fluorescence, luminescence, cell viability, relief of a cell nutritionalrequirement, cell growth, and drug resistance. For example, the reportergene can encode a gene product selected from the group consisting ofchloramphenicol acetyl transferase, luciferase, beta-galactosidase andalkaline phosphatase.

Also within the scope of the invention are methods for treating diseasesor disorders which are associated with an aberrant EFEMP1 level oractivity or which can benefit from modulation of the activity or levelof EFEMP1 (e.g. a macular degeneration). The methods compriseadministering, e.g., either locally or systemically to a subject, apharmaceutically effective amount of a composition comprising an EFEMP1therapeutic (e.g. an MD therapeutic).

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a fundus photograph of a patient affected with malattialeventinese (ML). The confluent yellow drusen in the center of thephotograph are characteristic of both ML and Doyne honeycomb retinaldystrophy, while the streak-like radial drusen in the periphery of thephotograph are the distinguishing feature of ML.

FIG. 2 depicts bacterial and yeast artificial chromosomes comprising theminimum tiling path of the EFEMP1 locus. The genetic markers andsequence tagged sites found to be present on each artificial chromosomeare shown as solid squares (on the YACs) or circles (on the BACs). Opencircles or squares indicate PCR failure. Disease intervals based onrecombination within families and shared haplotypes between families areindicated with brackets. The names of genes and expressed sequencetagged sites that were screened for coding sequence mutations are shownin bold.

FIG. 3 consists of representative chromatograms generated by fluorescentdye-terminator sequencing of PCR products from two affected individuals,which reveal a C→T transition at the first nucleotide of codon 345,which would be expected to alter the amino acid at this position from anarginine to a tryptophan: a) heterozygous Arg345Trp mutation; b)homozygous Arg345Trp mutation; and c) normal control.

FIG. 4 a) is a Northern blot analysis of mouse EFEMP1 and β-actin geneexpression. One μg of mouse embryonic and adult tissue poly (A) mRNAwere sequentially hybridized with ³²P-labelled cDNA probes for EFEMP1(upper pane) and β-actin (lower panel).

FIG. 4 b) shows the results of an RT-PCR analysis of RNA extracted fromthe human eye. Primers were chosen from the EFEMP1 coding sequences suchthat the amplimer included portions of exons 11 and 12 and would be ofexpected size (236 bp) only if it was amplified from the cDNA. The PCRwas performed with cDNA prepared from RNA extracted from: lane 2-humanneurosensory retina; lane 3-a mixture of human RPE and choroid; and,lane 4-isolated (but non-cultured) human RPE cells from a human donor.For lane 5, the template for amplification was genomic DNA while forlane 6, no template was added. Lane 1 contains the 100 bp ladder.

FIG. 5 shows human EFEMP1 genomic sequence including 5′ and 3′untranslated regions (UTRs), complete sequences of intron 2, 4,and 10and partial sequences of introns 3, and 5-9.

FIG. 6 shows human EFEMP1 cDNA sequence and the amino acid sequence ofthe hEFEMP1 protein.

4. DETAILED DESCRIPTION

4.2 General

The instant invention is based on linkage studies that have mapped amacular degeneration causing gene to a region of human chromosome 2 andon sequencing studies that have identified a mutation in the EFEMP1 genewithin the mapped region, that is associated with ML and DHRD. Thecoding sequence of the EFEMP1 gene is comprised of 1617 base pairs (FIG.5) and encodes a 539 amino acid extracellular matrix protein (SEQ ID NO1), which is likely to cause the accumulation of lipofuscin-likematerial under the retinal pigment epithelium in structures known asdrusen (the hallmark of MD). The finding that mutations in EFEMP1 causemacular degeneration allows for diagnostic testing for maculardegeneration on presymptomatic individuals, who are at risk ofdeveloping macular degeneration based on family history. In addition,tests can be performed on postsymptomatic individuals diagnosed withmacular degeneration based on an ophthalologic examination.

In addition to being used diagnostically, identification of theinvolvement of mutations in the EFEMP1 gene in the development ofmacular degeneration allows the production of cell-free and cell-basedscreening assays and transgenic animals for use in further studies ofthe disorder and to identify safe and effective MD therapeutics.

The identification of a single gene responsible for ML and DHRD can alsoimprove understanding of the types and classes of genes that can causerelated disorders. In addition, the identification of one gene productcausing a disorder can make it possible to identify other genes whichcan cause a similar phenotype. For example, the identification of thedystrophin gene has led to the isolation of dystrophin relatedglycoproteins, at least one of which plays a role in other forms ofmuscular dystrophy. Also, a gene capable of causing a Mendeliandisorder, may contribute to the inheritance of a multifactorial form ofthe disorder. A striking example of this has been the identification ofgenes involved in various forms of cancer (e.g. colon cancer) bystudying familial forms of cancer (e.g. hereditary nonpolyposis coloncancer and familial adenomatous polyposis). Groden, J. A. et al.,(1991)Cell 66:589-600; Aaltonen, L. A. (1993) Science 260:812-816). Forexample, as shown herein, AMD appears to be allelic to Doyne's maculardystrophy

4.2 Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “an aberrant activity”, as applied to an activity of apolypeptide such as, EFEMP1 refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polyeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart. Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide or polypeptide. A cell can have an aberrant EFEMP1 activity dueto overexpression or underexpression of a wild-type or mutant EFEMP1polypeptide.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably for the purposes herein, meansan effector or antigenic function that is directly or indirectlyperformed by an EFEMP1 polypeptide (whether in its native or denaturedconformation), or by any subsequence thereof. Biological activitiesinclude binding to a target peptide. An EFEMP1 bioactivity can bemodulated by directly affecting the binding between an EFEMP1 and anEFEMP1 binding partner. Alternatively, an EFEMP1 bioactivity can bemodulated by modulating the level of an EFEMP1 polypeptide, such as bymodulating expression of an EFEMP1 gene.

As used herein, the term “bioactive fragment of an EFEMP1 polypeptide”refers to a fragment of a full-length EFEMP1 polypeptide, wherein thefragment specifically mimics or antagonizes the activity of a wild-typeEFEMP1 polypeptide. The bioactive fragment preferably is a fragmentcapable of interacting with an EFEMP1 binding partner.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding one of the subject polypeptides with a secondamino acid sequence defining a domain (e.g. polypeptide portion) foreignto and not substantially homologous with any domain of one of thepolypeptides. A chimeric protein may present a foreign domain which isfound (albeit in a different protein) in an organism which alsoexpresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms.

“Complementary” sequences as used herein refer to sequences which havesufficient complementarity to be able to hybridize, forming a stableduplex.

The terms “control” or “control sample” refer to any sample appropriateto the detection technique employed. The control sample may contain theproducts of the allele detection technique employed or the material tobe tested. Further, the controls may be positive or negative controls.By way of example, where the allele detection technique is PCRamplification, followed by size fractionation, the control sample maycomprise DNA fragments of an appropriate size. Likewise, where theallele detection technique involves detection of a mutated protein, thecontrol sample may comprise a sample of a mutant protein. However, it ispreferred that the control sample comprises the material to be tested.

The phrases “disruption of the gene” and “targeted disruption” or anysimilar phrase refers to the site specific interruption of a native DNAsequence so as to prevent expression of that gene in the cell ascompared to the wild-type copy of the gene. The interruption maybecaused by deletions, insertions or modifications to the gene or anycombination thereof.

A “delivery complex” shall mean a targeting means (e.g. a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular uptake by atarget cell). Examples of targeting means include: sterols (e.g.cholesterol), lipids (e.g. a cationic lipid, virosome or liposome),viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) ortarget cell specific binding agents (e.g. ligands recognized by targetcell specific receptors). Preferred complexes are sufficiently stable invivo to prevent significant uncoupling prior to internalization by thetarget cell. However, the complex is cleavable under appropriateconditions within the cell so that the gene, protein, polypeptide orpeptide is released in a functional form.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding a polypeptide” may thus refer to one or more geneswithin a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a protein with the same biological activity.

An “EFEMP1” gene or protein refers to an “EGF-containing fibrillin-likeextracellular matrix protein 1 gene or protein. cDNA encoding a portionof the protein is posted in GenBank under accession number UO3877. Theacronym “EFEMP1” includes genes, proteins and portions thereof, whichare substantially homologous in structure and function, includingfibulin (1 and 2), Fibrillin, nidogen, notch, protein S and Factor IX.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame encoding one ofthe polypeptides of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid molecule encoding a polypeptide and comprising protein-encodingexon sequences, though it may optionally include intron sequences whichare derived from a chromosomal gene. Exemplary recombinant genesencoding the subject polypeptides are represented in the appendedSequence Listing. The term “intron” refers to a DNA sequence present ina given gene which is not translated into protein and is generally foundbetween exons.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present invention.

“Increased risk” refers to a statistically higher frequency ofoccurrence of the disease or condition in an individual carrying aparticular polymorphic allele in comparison to the frequency ofoccurrence of the disease or condition in a member of a population thatdoes not carry the particular polymorphic allele.

The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may beprotein-protein or protein-nucleic acid in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject polypeptides preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the gene ingenomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein-also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors nor otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

A “knock-in” transgenic animal refers to an animal that has had amodified gene introduced into its genome and the modified gene can be ofexogenous or endogenous origin.

A “knock-out” transgenic animal refers to an animal in which there ispartial or complete suppression of the expression of an endogenous gene(e.g, based on deletion of at least a portion of the gene, replacementof at least a portion of the gene with a second sequence, introductionof stop codons, the mutation of bases encoding critical amino acids, orthe removal of an intron junction, etc.)

A “knock-out construct” refers to a nucleic acid sequence that can beused to decrease or suppress expression of a protein encoded byendogenous DNA sequences in a cell.

“Linkage disequilibrium” refers to co-inheritance of two alleles atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given control population. The expectedfrequency of occurrence of two alleles that are inherited independentlyis the frequency of the first allele multiplied by the frequency of thesecond allele. As used herein, the term “linkage disequilibrium” alsorefers to linked sequences. Alleles that co-occur at expectedfrequencies are said to be in “linkage equilibrium” or “not linked.”When referring to allelic patterns that are comprised of more than oneallele, a first allelic pattern is in linkage disequilibrium with asecond allelic pattern if all the alleles that comprise the firstallelic pattern are in linkage disequilibrium with at least one of thealleles of the second allelic pattern.

“MD” or “Macular Degeneration” is a clinical term that is used todescribe a variety of diseases that are all characterized by aprogressive loss of central vision associated with abnormalities ofBruch's membrane and the retinal pigment epithelium. These disordersinclude very common conditions that affect older patients (age relatedmacular degeneration or AMD) as well as rarer, earlier-onset dystrophiesthat in some cases can be detected in the first few decades of life.Examples include Malattia Leventinese (ML) and Doyne's Honeycomb RetinalDystrophy (DHRD).

An “MD therapeutic” refers to an agent that is usefull in treating orpreventing the development of a Macular Degeneration. Examples includegenes, proteins (e.g. glycosylated or unglycosylated protein, polypepideor protein) or other organic or inorganic molecules (e.g. smallmolecules) that interfere with or compensate for, the biochemical eventsthat are causative of MD.

A “mutated gene” or “mutation” or “functional mutation” refers to anallelic form of a gene, which is capable of altering the phenotype of asubject having the mutated gene relative to a subject which does nothave the mutated gene. The altered phenotype caused by a mutation can becorrected or compensated for by certain agents. If a subject must behomozygous for this mutation to have an altered phenotype, the mutationis said to be recessive. If one copy of the mutated gene is sufficientto alter the phenotype of the subject, the mutation is said to bedominate. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene) the mutation is said to beco-dominant.

The “non-human animals” of the invention include mammalians such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse. The term“chimeric animal” is used herein to refer to animals in which therecombinant gene is found, or in which the recombinant gene is expressedin some but not all cells of the animal. The term “tissue-specificchimeric animal” indicates that one of the recombinant genes is presentand/or expressed or disrupted in some tissues but not others.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term “promoter” means a DNA sequence that regulatesexpression of a selected DNA sequence operably linked to the promoter,and which effects expression of the selected DNA sequence in cells. Theterm encompasses “tissue specific” promoters, i.e. promoters, whicheffect expression of the selected DNA sequence only in specific cells(e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”,with respect to a recombinant gene, is meant to include within themeaning of “recombinant protein” those proteins having an amino acidsequence of a native protein, or an amino acid sequence similar theretowhich is generated by mutations including substitutions and deletions(including truncation) of a naturally occurring form of the protein.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,peptidomimetics, carbohydrates, lipids or other organic or inorganicmolecules.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 20, 30, 50, 100,150, 200, 300, 350, 400 or 425 consecutive nucleotides.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the recombinant genes isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of proteins.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a polypeptide or, inthe case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the protein is disrupted.

As used herein, the term “transgene” means a nucleic acid sequenceencoding, e.g., one of the polypeptides, or an antisense transcriptthereto, which is partly or entirely, heterologous, i.e., foreign, tothe transgenic animal or cell into which it is introduced, or, ishomologous to an endogenous gene of the transgenic animal or cell intowhich it is introduced, but which is designed to be inserted, or isinserted, into the animal's genome in such a way as to alter the genomeof the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout). A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, (e.g.as intron), that may be necessary for optimal expression of a selectednucleic acid.

A“transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. This,molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the proteins, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant gene issilent are also contemplated, as for example, the FLP or CRE recombinasedependent constructs described below. Moreover, “transgenic animal” alsoincludes those recombinant animals (“knockouts”) in which genedisruption of one or more genes is caused by human intervention,including both recombination and antisense techniques.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of a condition or disease.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

4.3. Nucleic Acids of the Present Invention

The invention provides EFEMP1 nucleic acids, homologs thereof, andportions thereof. Preferred nucleic acids have a sequence at leastabout, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, and more preferably 85%, 90%,95% homologous and more preferably 98% and even more preferably at least99% homologous with a nucleotide sequence of an EFEMP1 gene, e.g., suchas a sequence shown-in one of FIG. 5 or comnplement thereof. Inpreferred embodiments, the nucleic acid is mammalian and in particularlypreferred embodiments, includes all or a portion of the nucleotidesequence corresponding to the coding region of one of FIG. 5.

The invention also pertains to isolated nucleic acids comprising anucleotide sequence encoding EFEMP1 polypeptides, variants and/orequivalents of such nucleic acids. The term equivalent is understood toinclude nucleotide sequences encoding functionally equivalent EFEMP1polypeptides or functionally equivalent peptides having an activity ofan EFEMP1 protein such as described herein. Equivalent nucleotidesequences will include sequences that differ by one or more nucleotidesubstitution, addition or deletion, such as allelic variants; and will,therefore, include sequences that differ from the nucleotide sequence ofthe EFEMP1 gene shown in FIG. 5 due to the degeneracy of the geneticcode.

Preferred nucleic acids are vertebrate EFEMP1 nucleic acids.Particularly preferred vertebrate EFEMP1 nucleic acids are mammalian.Regardless of species, particularly preferred EFEMP1 nucleic acidsencode polypeptides that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%,80%, 90%, or 95% similar or idenitical to an amino acid sequence of avertebrate EFEMP1 protein. In one embodiment, the nucleic acid is a cDNAencoding a polypeptide having at least one bioactivity of the subjectEFEMP1 polypeptide. Preferably, the nucleic acid includes all or aportion of the nucleotide sequence corresponding to the nucleic acid ofFIG. 5.

Still other preferred nucleic acids of the present invention encode anEFEMP1 polypeptide which is comprised of at least 2, 5, 10, 25, 50, 100,150, 200, 250, 300, 350 or 400 amino acid residues. For example, suchnucleic acids can comprise about 50, 60, 70, 80, 90, or 100 base pairs.Also within the scope of the invention are nucleic acid molecules foruse as probes/primer or antisense molecules (i.e. noncoding nucleic acidmolecules), which can comprise at least about 6, 12, 20, 30, 50, 60, 70,80, 90 or 100 base pairs in length.

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid represented by FIG. 5 orcomplement thereof or the nucleic acids having ATCC Designation No.______. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or temperature and salt concentration may be heldconstant while the other variable is changed. In a preferred embodimentan MFGF nucleic acid of the present invention will bind to one of SEQ IDNOS. 2, 3, or 5 or the sequence shown in FIG. 5 or complement thereofunder moderately stringent conditions, for example at about 2.0×SSC andabout 40° C. In a particularly preferred embodiment, an MFGF nucleicacid of the present invention will bind to one of SEQ ID NOS. 2, 3, or5, or the sequence shown in FIG. 5 or complement thereof under highstringency conditions.

Nucleic acids having a sequence that differs from the nucleotidesequences shown in FIG. 5 or complement thereof due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., peptides having abiological activity of an EFEMP1 polypeptide) but differ in sequencefrom the sequence shown in the sequence: listing due to degeneracy inthe genetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of anEFEMP1 polypeptide. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject EFEMP1 polypeptides will exist among mammals. One skilled in theart will appreciate that these variations in one or more nucleotides(e.g., up to about 3-5% of the nucleotides) of the nucleic acidsencoding polypeptides having an activity of an EFEMP1 polypeptide mayexist among individuals of a given species due to natural allelicvariation.

Nucleic acids of the invention can encode one or more domains of anEFEMP1 protein (e.g. the EGF domain). Other preferred nucleic acids ofthe invention include nucleic acids encoding derivatives of EFEMP1polypeptides which lack one or more biological activities of EFEMP1polypeptides. Such nucleic acids can be obtained, e.g., by a first roundof screening of libraries for the presence or absence of a firstactivity and a second round of screening for the presence or absence ofanother activity.

Also within the scope of the invention are nucleic acids encoding splicevariants or nucleic acids representing transcripts synthesized from analternative transcriptional initiation site, such as those whosetranscription was initiated from a site in an intron.

In preferred embodiments, the EFEMP1 nucleic acids can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate pepidenucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.

Such modified nucleic acids can be used as antisense or antigene agentsfor sequence-specific modulation of gene expression or in the analysisof single base pair mutations in a gene by, e.g., PNA directed PCRclamping or as probes or primers for DNA sequence and hybridization(Hyrup B. et al (1996) supra; Perry-O'Keefe supra).

PNAs can further be modified, e.g., to enhance their stability orcellular uptake, e.g., by attaching lipophilic or other helper groups tothe PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. MFGFPNAs can also be linked to DNA as described, e.g., in Hyrup B. (1996)supra and Finn P. J. et al. (1996) Nucleic Acids Research 24 (17):3357-63. For example, a DNA chain can be synthesized on a solid supportusing standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag,M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNAsegment and a 3′ DNA segment (Finn P. J. et al. (1996) supra).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser, K. H. et al. (1975) BioorganicMed Chem. Lett. 5: 1119-11124).

In other embodiments, EFEMP1 nucleic acids may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents that facilitate transport across the cell membrane.

4.3.1 Probes and Primers

The nucleotide sequences determined from the cloning of EFEMP1 genesfrom mammalian organisms will further allow for the generation of probesand primers designed for use in identifying and/or cloning EFEMP1homologs in other cell types, e.g., from other tissues, as well asEFEMP1 homologs from other mammalian organisms. For instance, thepresent invention also provides a probe/primer comprising asubstantially purified oligonucleotide, which oligonucleotide comprisesa region of nucleotide sequence that hybridizes understringentconditions to at least approximately 12, preferably 25, more preferably40, 50 or 75 consecutive nucleotides of sense or anti-sense sequenceselected from FIG. 5 or naturally occurring mutants thereof. Forinstance, primers based on the nucleic acid represented in FIG. 5 can beused in PCR reactions to clone EFEMP1 homologs.

Likewise, probes based on the subject EFEMP1 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins, for use, e.g, in prognostic or diagnostic assays (furtherdescribed below). In preferred embodiments, the probe further comprisesa label group attached thereto and able to be detected, e.g., the labelgroup is selected from amongst radioisotopes, fluorescent compounds,enzymes, and enzyme co-factors.

Probes and primers can be prepared and modified, e.g., as previouslydescribed herein for other types of nucleic acids.

4.3.2 Antisense, Ribozyme and Triplex Techniques

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g., bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject EFEMP1 proteins so as to inhibitexpression of that protein, e.g., by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an EFEMP1 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of anEFEMP1 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the EFEMP1 nucleotide sequence of interest, arepreferred.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to EFEMP1 mRNA. The antisenseoligonucleotides will bind to the EFEMP1 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofan EFEMP1 gene could be used in an antisense approach to inhibittranslation of endogenous EFEMP1 mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but couldalso be used in accordance with the invention. Whether designed tohybridize to the 5′, 3′ or coding region of EFEMP1 mRNA, antisensenucleic acids should be at least six nucleotides in length, and arepreferably less than about 100 and more preferably less than about 50,25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. W088/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. W089/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxytiethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-gomersand are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl.Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.One advantage of PNA oligomers is their ability to bind to complementaryDNA essentially independently from the ionic strength of the medium dueto the neutral backbone of the DNA. In yet another embodiment, theantisense oligonucleotide comprises at least one modified phosphatebackbone selected from the group consisting of a phoshorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate olgonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988,Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the EFEMP1 coding regionsequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

The antisense molecules can be delivered to cells which express EFEMP1in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense sufficient to suppress translation on endogenous mRNAs incertain instances. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willfor complementary base pair's with the endogenous EFEMP1 transcripts andthereby prevent translation of the EFEMP1 mRNA. For example, a vectorcan be introduced in vivo such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA, technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive and can include but not be limited to: the SV40 earlypromoter region (Bernoist and Chambon, 1981, Nature 290:304-310), thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which: can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave EFEMP1 mRNAtranscripts can also be used to prevent translation of EFEMP1 mRNA andexpression of EFEMP1 (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroyEFEMP1 mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach, 1988, Nature, 334:585-591. There are a number of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofhuman EFEMP1 cDNA. Preferably the ribozyme is engineered so that thecleavage recognition site is-located near the 5′ end of the EFEMP1 mRNA;i.e., to increase efficiency and minimize the intracellular accumulationof non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984 Science, 224:574-578; Zaug andCech, 1986; Science, 231 470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO88/04300by University Patents Inc., Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in EFEMP1 gene.

As in the antisense approach, the ribozyrnes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the EFEMP1 gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous EFEMP1 messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Endogenous EFEMP1 gene expression can also be reduced by inactivating or“knocking out” the EFEMP1 gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional EFEMP1 (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenousEFEMP1 gene (either the coding regions or regulatory regions of theEFEMP1 gene) can be used, with or without a selectable marker and/or anegative selectable marker, to transfect cells that express EFEMP1 invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the EFEMP1 gene. Suchapproaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive EFEMP1 (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be adapted foruse in humans provided the recombinant DNA constructs are directlyadministered or targeted to the required site in vivo using appropriateviral vectors.

Alternatively, endogenous EFEMP1 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the EFEMP1 gene (i.e., the EFEMP1 promoter and/or enhancers)to form triple helical structures that prevent transcription of theEFEMP1 gene in target cells in the body. (See generally, Helene, C.1991, Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann.N.Y. Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimnidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on: a single strand of the targeted duplex, resulting in CGCtriplets, across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polyerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications, include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphcidiesteraselinkages within the oligodeoxyribonucleotide backbone.

4.3.3. Vectors Encoding MFGF Proteins and MFGF Expressing Cells

The invention further provides plasmids and vectors encoding an EFEMP1protein, which can be used to express an EFEMP1 protein in a host cell.The host cell may be any prokaryotic or eukaryotic cell. Thus, anucleotide sequence derived from the cloning of mammalian EFEMP1proteins, encoding all or a selected portion of the full-length protein,can be used to produce a recombinant form of an EFEMP1 polypeptide viamicrobial or eukaryotic cellular process. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial) cells, arestandard procedures well known in the art.

Vectors that allow expression of a nucleic acid in a cell are referredto as expression vectors. Typically, expression vectors used forexpressing an EFEMP1 protein contain a nucleic acid encoding an EFEMP1polypeptide, operably linked to at least one transcriptional regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the subject EFEMP1 proteins. Transcriptionalregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). In one embodiment, the expression vector includes a recombinantgene encoding a peptide having an agonistic activity of a subject EFEMP1polypeptide, or alternatively, encoding a peptide which is anantagonistic form of an EFEMP1 protein.

Suitable vectors for the expression of an EFEMP1 polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an MFGF polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the EFEMP1 genes represented in FIG. 5.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant EFEMP1polypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III)

When it is desirable to express only a portion of an EFEMP1. protein,such as a form lacking a portion of the N-terminus, i.e. a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing MFGF derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae ), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Moreover, the gene constructs of the present invention can also be usedas part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject EFEMP1proteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of an EFEMP1polypeptide in particular cell types so as to reconstitute the functionof, or altematively, abrogate function of EFEMP1 in a tissue. This couldbe desirable, for example, when the naturally-occurring form of theprotein is misexpressed or the natural protein is mutated and lessactive.

In addition to viral transfer methods, non-viral methods can also beemployed to cause expression of a subject EFEMP1 polypeptide in thetissue of an animal. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral targeting means of the present invention rely on endocyticpathways for the uptake of the subject EFEMP1 polypeptide gene by thetargeted cell. Exemplary targeting means of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

In other embodiments transgenic animals, described in more detail belowcould be used to produce recombinant proteins.

4.4. Polypeptides of the Present Invention

The present invention makes available isolated EFEMP1 polypeptides whichare isolated from, or otherwise substantially free of other cellularproteins. The term “substantially free of other cellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations ofEFEMP1 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein.

Preferred EFEMP1 proteins of the invention have an amino acid sequencewhich is at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, or 95% identical orhomologous to an amino acid sequence of SEQ ID NO. 1. Even morepreferred EFEMP1 proteins comprise an amino acid sequence which is atleast about 97, 98, or 99% homologous or identical to an amino acidsequence of SEQ ID NOS. 1. Such proteins can be recombinant proteins,and can be, e.g., produced in vitro from nucleic acids comprising anucleotide. sequence set forth in FIG. 5 or homologs thereof Forexample, recombinant polypeptides preferred by the present invention canbe encoded by a nucleic acid, which is at least 85% homologous and morepreferably 90% homologous and most preferably 95% homologous with anucleotide sequence set forth in FIG. 5. Polypeptides which are encodedby a nucleic acid that is at least about 98-99% homologous with thesequence of FIG. 5 are also within the scope of the invention.

In a preferred embodiment, an EFEMP1 protein of the present invention isa mammalian EFEMP1 protein. In a particularly preferred embodiment anEFEMP1 protein is set forth as SEQ ID NO. 1. In particularly preferredembodiments, an EFEMP1 protein has an EFEMP1 bioactivity. It will beunderstood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the EFEMP1 protein relative to the unmodified polypeptide chain.

The invention also features protein isoforms encoded by splice variantsof the present invention. Such isoforms may have biological activitiesidentical to or different from those possessed by the EFEMP1 proteinspecified by SEQ ID NO. 1.

EFEMP1 polypeptides preferably are capable of functioning as either anagonist or antagonist of at least one biological activity of a wild-type(“authentic”) EFEMP1 protein of the appended sequence listing.

Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75 and 100, amino acids in length are within thescope of the present invention.

For example, isolated EFEMP1 polypeptides can be encoded by all or aportion of a nucleic acid sequence shown in any of SEQ ID NO. 1.Isolated peptidyl portions of EFEMP1 proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, an EFEMP1 polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) EFEMP1 protein. Assays fordetermining whether a compound, e.g, a protein, such as an EFEMP1protein or variant thereof, has one or more of the above biologicalactivities are well known in the art.

Other preferred proteins of the invention are fusion proteins, e.g.,EFEMP1-immunoglobulin fusion proteins. Such fusion proteins can provide,e.g., enhanced stability and solubility of EFEMP1 proteins and may thusbe useful in therapy. Fusion proteins can also be used to produce animmunogenic fragment of an EFEMP1 protein. For example, the VP6 capsidprotein of rotavirus can be used as an immunologic carrier protein forportions of the EFEMP1 polypeptide, either in the monomeric form or inthe form of a viral particle. The nucleic acid sequences correspondingto the, portion of a subject EFEMP1 protein to which antibodies are tobe raised can be incorporated into a fusion gene construct whichincludes coding sequences for a late vaccinia virus structural proteinto produce a set of recombinant viruses expressing fusion proteinscomprising MFGF epitopes as part of the virion. It has, beendemonstrated with the use of immunogenic fusion proteins utilizing theHepatitis B surface antigen fusion proteins that recombinant Hepatitis Bvirions can be utilized in this role as well. Similarly, chimericconstructs coding for fusion proteins containing a portion of an EFEMP1protein and the poliovirus capsid protein can be created to enhanceimmunogenicity of the set of polypeptide antigens (see, for example, EPPublication No: 0259149; and Evans et al. (1989) Nature 339:385; Huanget al. (1988) J. Virol. 62:3855; and Schlienger et al. (1992) J. Virol.66:2).

The Multiple antigen peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofan EFEMP1 polypeptide is obtained directly from organo-chemicalsynthesis of the peptide onto an oligomeric branching lysine core (see,for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al.(1992) J. Immunol. 148:914). Antigenic determinants of EFEMP1 proteinscan also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the EFEMP1 polypeptides of the present invention. For example, EFEMP1polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theEFEMP1 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

The present invention further pertains to methods of producing thesubject EFEMP1 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant EFEMP1polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant EFEMP1 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject EFEMP1 polypeptides which function in a limited capacity as oneof either an EFEMP1 agonist (mimetic) or an EFEMP1 antagonist, in orderto promote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of EFEMP1 proteins.

Homologs of each of the subject EFEMP1 proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the EFEMP1 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an EFEMP1 receptor.

The recombinant EFEMP1 polypeptides of the present invention alsoinclude homologs of the wildtype EFEMP1 proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter ubiquitination or other enzymatictargeting associated with the protein.

EFEMP1 polypeptides may also be chemically modified to create EFEMP1derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of EFEMP1 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

Modification of the structure of the subject EFEMP1 polypeptides can befor such purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e.g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the EFEMP1 polypeptides describedin more detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional MFGF homolog (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject EFEMP1 proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g., homologs). The purpose of screening suchcombinatorial libraries is to generate, for example, novel EFEMP1homologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein.

In one embodiment, the variegated library of EFEMP1 variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential EFEMP1 sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofEFEMP1 sequences therein.

There are many ways by which such libraries of potential EFEMP1 homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential EFEMP1 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc3^(rd) Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Likewise, a library of coding sequence fragments can be provided for anEFEMP1 clone in order to generate a variegated population of EFEMP1fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of an EFEMP1 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of EFEMP1 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate EFEMP1 sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 10²⁶ molecules. Combinatorial libraries of this size maybetechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-331).

The invention also provides for reduction of the EFEMP1 proteins togenerate mimetics, e.g., peptide or non-peptide agents, such as smallmolecules, which are able to disrupt binding of an EFEMP1 polypeptide ofthe present invention with a molecule, e.g. target peptide. Thus, suchmutagenic techniques as described above are also useful to map thedeterminants of the EFEMP1 proteins which participate in protein-proteininteractions involved in, for example, binding of the subject EFEMP1polypeptide to a target peptide. To illustrate, the critical residues ofa subject EFEMP1 polypeptide which are involved in molecular recognitionof its receptor can be determined and used to generate EFEMP1 derivedpeptidomimetics or small molecules which competitively inhibit bindingof the authentic EFEMP1 protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of thesubject EFEMP1 proteins which are involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residues ofthe EFEMP1 protein which facilitate the interaction. Such mimetics maythen be used to interfere with the normal function of an EFEMP1 protein.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9^(th) American Peptide Symposium)Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagaiet al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem SocPerkin Trans 1:1231), and b-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71).

4.5. Anti-EFEMP1 Antibodies and Uses Therefor

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian EFEMP1 protein, e.g., a wild-type or mutatedEFEMP1 protein. For example, by using immunogens derived from an EFEMP1protein, e.g., based on the cDNA sequences, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of thepeptide (e.g., a mammalian EFEMP1 polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion proteinas described above). Techniques for conferring immunogenicity on aprotein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of an EFEMP1 protein canbe administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers plasma orserum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies. In a preferredembodiment, the subject antibodies are immunospecific for antigenicdeterminants of an EFEMP1 protein of a mammal, e.g., antigenicdeterminants of a protein set forth in SEQ ID No: 1 or closely relatedhomologs (e.g., at least 90% homologous, and more preferably at least94% homologous).

Following immunization of an animal with an antigenic preparation of anEFEMP1 polypeptide, anti-EFEMP1 antisera can be obtained and, ifdesired, polyclonal anti-EFEMP1 antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells. (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique originally developed byKohler and Milstein ((1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian EFEMP1polypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human EFEMP1 antibodies specifically react with the protein encodedby a nucleic acid having a sequence shown in FIG. 5.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian EFEMP1 polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific single-chain, and chimeric and humanizedmolecules having affinity for an EFEMP1 protein conferred by at leastone CDR region of the antibody. In preferred embodiments, the antibodyfurther comprises a label attached thereto and able to be detected,(e.g., the label can be a radioisotope, fluorescent compound, enzyme orenzyme co-factor).

Anti-EFEMP1 antibodies can be used, e.g., to monitor EFEMP1 proteinlevels in an individual for determining, e.g., whether a subject has adisease or condition associated with an aberrant EFEMP1 protein level,or allowing determination of the efficacy of a given treatment regimenfor an individual afflicted with such a disorder. The level of EFEMP1polypeptides may be measured from cells in bodily fluid, such as inblood samples.

Another application of anti-EFEMP1 antibodies of the present inventionis in the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of an EFEMP1protein, e.g., other orthologs of a particular EFEMP1 protein or otherparalogs from the same species, can then be detected with antibodies,as, for example, reacting nitrocellulose filters lifted from infectedplates with anti-EFEMP1 antibodies. Positive phage detected by thisassay can then be isolated from the infected plate. Thus, the presenceof EFEMP1 homologs can be detected and cloned from other animals, as canalternate isoforms (including splice variants) from humans.

4.6 Predictive Medicine

4.6.1. MD Causative Mutations

The invention is based, at least in part, on the identification ofmutations that cause Macular Degeneration (MD). Because the particularMD mutations may be in linkage disequilibrium with other alleles, thedetection of such other alleles can also indicate a predisposition todeveloping MD in a subject.

4.6.2. Detection of Alleles

Many methods are available for detecting specific alleles at humanpolymorphic loci. The preferred method for detecting a specificpolymorphic allele may depend, in part, upon the molecular nature of thepolymorphism. For example, detection of specific alleles may be nucleicacid techniques based on hybridization, size, or sequence, such asrestriction fragment length polymorphism (RFLP), nucleic acidsequencing, and allele specific oligonucleotide (ASO) hybridization. Inone embodiment, the methods comprise detecting in a sample of DNAobtained from a subject the existence of an allele associated with MD.For example, a nucleic acid composition comprising a nucleic acid probeincluding a region of nucleotide sequence which is capable ofhybridizing to a sense or antisense sequence to an allele associatedwith MD can be used as follows: the nucleic acid in a sample is renderedaccessible for hybridization, the, probe is contacted with the nucleicacid of the sample, and the hybridization of the probe to the samplenucleic acid is detected. Such technique can be used to detectalterations or allelic variants at either the genomic or mRNA level aswell as to determine mRNA transcript levels, when appropriate.

A preferred detection method is ASO hybridization using probesoverlapping an allele associated with MD and has about 5, 10, 20, 25, or30 nucleotides around the mutation or polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to other allelic variants involved in MD are attached to asolid phase support, e.g., a “chip” (which can hold up to about 250,000oligonucleotides). Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. Mutation detection analysisusing these chips comprising oligonucleotides, also termed “DNA probearrays” is described e.g., in Cronin et al., Human Mutation 7:244, 1996.In one embodiment, a chip comprises all the allelic vanants of at leastone polymorphic region of a gene. The solid phase support is thencontacted with a test nucleic acid and hybridization to the specificprobes is detected. Accordingly, the identity of numerous allelicvariants of one or more genes can be identified in a simplehybridization experiment.

These techniques may also comprise the step of amplifying the nucleicacid before analysis. Amplification techniques are known to those ofskill in the art and include, but are not limited to cloning, polymerasechain reaction (PCR), polymerase chain reaction of specific alleles(ASA), ligase chain reaction (LCR), nested polymerase chain reaction,self sustained sequence replication (Guatelli, J. C. et al., Proc. Natl.Acad. Sci. USA 87:1874-78, 1990), transcriptional amplification system(Kwoh, D. Y. et al., Proc. Natl. Acad. Sci. USA 86:1173-77, 1989), andQ-Beta Replicase (Lizardi, P. M. et al., Bio/Technology 6:1197, 1988).

Amplification products may be assayed in a variety of ways, includingsize analysis, restriction digestion followed by size analysis,detecting specific tagged oligonucleotide primers in the reactionproducts, ASO hybridization, allele specific 5′ exonuclease detection,sequencing, hybridization, and the like.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that have detectablelabels that are different and thus can each be differentially detected.Of course, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

In a merely illustrative embodiment, the method includes the steps of(i) collecting a sample of cells from a patient, (ii) isolating nucleicacid (e.g., genomric, RNA or both) from the cells of the sample, (iii)contacting the nucleic acid sample with one or more primers whichspecifically hybridize to an allele associated with MD, under conditionssuch that hybridization and amplification of the desired marker occurs,and (iv) detecting the amplification product. These detection schemesare especially useful for the detection of nucleic acid molecules ifsuch molecules are present in very low numbers.

An allele associated with MD can also be identified by alterations inrestriction enzyme cleavage patterns through RFLP analysis. For example,sample and control DNA is isolated, amplified (optionally), digestedwith one or more restriction endonucleases, and fragment length sizesare determined by gel electrophoresis through size fractionization.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence a polymorphic sitehaving at least one allele associated with MD. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl. Acad. Sci. USA 74:560, 1977) or Sanger (Sanger etal., Proc. Nat. Acad. Sci. USA 74:5463, 1977). It is also contemplatedthat any of a variety of automated sequencing procedures may be utilizedwhen performing the subject assays (Biotechniques 19:448, 1995),including sequencing by mass spectrometry (see, for example PCTpublication WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-62, 1996;and Griffin et al., Appl Biochem. Biotechnol 38:147-59, 1993). It willbe evident to one skilled in the art that, for certain embodiments, theoccurrence of only one, two or three of the nucleic acid bases need bedetermined in the sequencing reaction. For instance, A-track or thelike, e.g., where only one nucleic acid is detected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers et al., Science 230:1242, 1985). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either, DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. (See,for example, Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397, 1988;Saleeba et al., Methods Enzymol. 217:286-95, 1992) In a preferredembodiment, the control DNA or RNA can have a detectable label.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes). For example, the mutYenzyme of E. coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.,Carcinogenesis 15:1657-62, 1994). According to an exemplary embodiment,an appropriate probe is hybridized to a cDNA or other DNA product from atest cell(s). The duplex is treated with a DNA mismatch repair enzyme,and the cleavage products, if any, can be detected from electrophoresisprotocols or the like. (See, for example, U.S. Pat. No. 5,459,039.)

In other embodiments, alterations in electrophoretic mobility will beused to identify an allele associated with MD. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, 1989,see also Cotton, Mutat. Res. 285:125-44, 1993; and Hayashi, Genet. Anal.Tech. Appl. 9:73-79, 1992. Single-stranded DNA fragments of sample andcontrol are denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes, such as primers with a detectable label.The sensitivity of the assay may be enhanced by using RNA (rather thanDNA), in which the secondary structure is more sensitive to a change insequence. In a preferred embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility (Keen et al., TrendsGenet. 7:5, 1991).

In yet another embodiment, the movement of an allele associated with MDin polyacrylamnide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.,Nature 313:495, 1985). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradientissued in place of a denaturing agent gradient to identify differencesin the mobility of control and sample DNA (Rosenbaum and Reissner,Biophys. Chem. 265:12753, 1987).

Examples of other techniques for detecting alleles associated with MDinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation or nucleotide difference (e.g., in allelic variants) is placedcentrally and then hybridized to target DNA under conditions whichpermit hybridization only if a perfect match is found (Saiki et al.,Nature 324:163, 1986); Saiki et al., Proc. Natl. Acad. Sci. USA 86:6230,1989). Such ASO hybridization techniques may be used to test onemutation or polymorphic region per reaction when oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations or polymorphic regions when the oligonucleotides are attachedto the hybridizing membrane and hybridized with labelled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation or polymorphic region of interest in the centerof the molecule (so that amplification depends on differentialhybridization) (Gibbs et al., Nucleic Acids Res. 17:2437-2448, 1989) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner, Tibtech11:238, 1993. In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al., Mol. Cell Probes 6:1, 1992). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany, Proc. Natl. Acad.Sci USA 88:189, 1991). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren et al., Science 241:1077-80,1988. The OLA protocol uses two oligonucleotides which are designed tobe capable of hybridizing to abutting sequences of a single strand of atarget. One of the oligonucleotides is linked to a separation marker,e.g,. biotinylated, and the other has a detectable label. If the precisecomplementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate. Ligation then permits the labeled oligonucleotideto be recovered using avidin, or another biotin ligand. Nickerson, D. A.et al. have described a nucleic acid detection assay that combinesattributes of PCR and OLA (Nickerson et al., Proc. Natl. Acad. Sci. USA87:8923-27, 1990. In this method, PCR is used to achieve the exponentialamplification of target DNA, which is then detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect alleles associated with MD. For example, U.S. Pat. No.5,593,826 discloses an OLA using an oligonucleotide having 3′-aminogroup and a 5′-phosphorylated oligonucleotide to form a conjugate havinga phosphoramidate linkage. In another variation of OLA described in Tobeet al., Nucleic Acids Res. 24:3728, 1996, OLA combined with PCR permitstyping of two alleles in a single microtiter well. By marking each ofthe allele-specific primers with a unique hapten, i.e. digoxigenin andfluorescein, each OLA reaction can be detected by using hapten specificantibodies that are labeled with different enzyme reporters, alkalinephosphatase or horseradish peroxidase. This system permits the detectionof the two alleles using a high throughput format that leads to theproduction of two different colors.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in U.S. Pat. No.4,656,127 (Mundy et al.). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.French Patent 2,650,840; PCT, Appln. No. WO91/02087. As in the Mundymethod of U.S. Pat. No. 4,656,127, a primer is employed that iscomplementary to allelic sequences immediately 3′ to a polymorphic site.The method determines the identity of the nucleotide of that site usinglabeled dideoxynucleotide derivatives, which, if complementary to thenucleotide of the polymorphic site will become incorporated onto theterminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet et al. in PCT Appln. No. 92/15712. The method ofGoelet et al. uses mixtures of labeled terminators and a primer that iscomplementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.,French Patent 2,650,840 and PCT Appln. No. WO91/02087, the method ofGoelet et al. is preferably a heterogeneous phase assay, in which theprimer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide inccorporation procedures forassaying polymorphic sites in DNA have been described (Komher et al.,Nucleic Acids Res. 17:7779-84, 1989; Sokolov, Nucleic Acids Res.18:3671, 1990; Syvanen et al., Genomics 8:684-92, 1990; Kuppuswamy etal., Proc. Natl. Acad. Sci. USA 88:1143-47, 1991; Prezant et al., Hum.Mutat. 1:159-64, 1992; Ugozzoli et al., GATA 9:107-12, 1992; Nyren etal., Anal. Biochem. 208:171-75, 1993). These methods differ from GBA™ inthat they all rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, et al., Amer. J. Hum. Genet. 52:46-59, 1993).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest et. al., Hum. Mol. Genet. 2:1719-21, 1993; van der Luijtet. al., Genomics 20:1-4, 1994). For PTT, RNA is initially isolated fromavailable tissue and reverse-transcribed, and the segment of interest isamplified by PCR. The products of reverse transcription PCR are thenused as a template for nested PCR amplification with a primer thatcontains an RNA polymerase promoter and a sequence for mitiatingeukaryotic translation. After amplification of the region of interest,the unique motifs incorporated into the primer permit sequential invitro transcription and translation of the PCR products. Upon sodiumdodecyl sulfate-polyacrylamide gel electrophoresis of translationproducts, the appearance of truncated polypeptides signals the presenceof a mutation that causes premature termination of translation. In avariation of this technique, DNA (as opposed to RNA) is used as a PCRtemplate when the target region of interest is derived from a singleexon.

In still another method known as Dynamic Allele Specific Hybridization(DASH), a target sequence is amplified by PCR in which one primer isbiotinylated. The biotinylated product strand is bound to a streptavidinor avidin coated microtiter plate well, and the non-biotinylated strandis rinsed away with alkali. An oligonucleotide probe, specific for oneallele, is hybridized to the target at low temperature. This forms aduplex DNA region that interacts with a double strand-specificintercalating dye. Upon excitation, the dye emits fluorescenceproportional to the amount of double stranded DNA probe-target duplex)present. The sample is then steadily heated while fluorescence iscontinually monitored. A rapid fall in fluorescence indicates thedenaturing (or “melting”) temperature of the probe-target duplex. Whenperformed under appropriate buffer and dye conditions, a single-basemismatch between the probe and the target results in a dramatic loweringof melting temperature (Tm) that can be easily detected (Howell, W. M.et al., (1999) Nature Biotechnology 17:)87-88.

Any cell type or tissue may be utilized in the diagnostics describedherein. In a preferred embodiment the DNA sample is obtained from abodily fluid obtained by known techniques. Alternatively, nucleic acidtests can be performed on dry samples (e.g. hair or skin).

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, PCR in situ Hybridization:Protocols and Applications (Raven Press, N.Y., 1992)).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Another embodiment of the invention is directed to kits. This kit maycontain one or more oligonucleotides, including 5′ and 3′oligonucleotides that hybridize 5′ and 3′ to a polymorphic site havingas allele associated with MD or detection oligonucleotides thathybridize directly to an allele associate with MD. The kit may alsocontain one or more oligonucleotides capable of hybridizing near or atother alleles that are in linkage disequilibrium with an MD causingallele (mutation). PCR amplification oligonucleotides should hybridizebetween 25 and 2500 base pairs apart, preferably between about 100 andabout 500 bases apart, in order to produce a PCR product of convenientsize for subsequent analysis.

For use in a kit, oligonucleotides may be any of a variety of naturaland/or synthetic compositions such as synthetic oligonucleotides,restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs),and the like. The assay kit and method may also employ oligonucleotideshaving detectable labels to allow ease of identification in the assays.Examples of labels which may be employed include radio-labels, enzymes,fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties,metal binding moieties, antigen or antibody moieties, and the like.Oligonucleotides useful in kits as well as other aspects of the presentinvention are selected from the group consisting of any oligonucleotidesthat overlap or are contained in SEQ. ID. Nos. 46-74. Particularlypreferred primers can be selected from any of SEQ ID Nos 2-43. One ofskill in the art can readily determine additional useful oligonucleotidesequences based on the sequences provided herein.

The kit may, optionally, also include DNA sampling means; DNApurification reagents such as Nucleon™ kits, lysis buffers, proteinasesolutions and the like; PCR reagents, such as 10× reaction buffers,thermostable polymerase, dNTPs, and the like; and DNA detection meanssuch as appropriate restriction enzymes, allele specificoligonucleotides, degenerate oligonucleotide primers for nested PCR.

4.6.3. Pharmacogenomics,

Knowledge of the particular MD associated mutations, alone or inconjunction with information on other genetic defects contributing to MD(the genetic profile of MD) allows a customization of the therapy to theindividual's genetic profile, the goal of “pharmaeogenomics”. Thus,comparison of a subject's particular genetic profile to the geneticprofile of MD, permits the selection or design of drugs that areexpected to be safe and efficacious for a particular patient or patientpopulation (i.e., a group of patients having the same geneticalteration).

The ability to target populations expected to show the highest clinicalbenefit, based on genetic profile, can enable: 1) the repositioning ofmarketed drugs with disappointing market results; 2) the rescue of drugcandidates whose clinical development has been discontinued as a resultof safety or efficacy limitations, which are patient subgroup-specific;and 3) an accelerated and less costly development for drug candidatesand more optimal drug labeling (e.g. since measuring the effect ofvarious doses of an agent on an MD causative mutation is useful foroptimizing effective dose).

Cells of a subject may also be obtained before and after administrationof a candidate MD therapeutic to detect the level of expression of genesother than EFEMP1, to verify that the therapeutic does not increase ordecrease the expression of genes which could be deleterious. This can bedone, e.g., by using the method of transcriptional profiling. Thus, mRNAfrom cells exposed in vivo to a therapeutic and mRNA from the same typeof cells that were not exposed to the therapeutic could be reversetranscribed and hybridized to a chip containing DNA from numerous genes,to thereby compare the expression of genes in cells treated and nottreated with the therapeutic.

4.7. EFEMP1 Bassed Therapeutics

4.7.1 EEMP1 Therapeutics

Agents that modulate an EFEMP1 bioactivity should prove useful intreating or preventing a number of EFEMP1 are useful in treating orpreventing the development of diseases such as macular degeneration.Such EFEMP1 therapeutics can comprise nucleic acids (e.g. genes,fragments thereof, antisense molecule, proteins (e.g. glycosylated orunglycosylated protein, polypeptide or protein) or other organic orinorganic molecules (e.g. small molecules) that interfere with orcompensate for the biochemical events that are causative of MD. Thefollowing describes in vitro and in vivo assays for identifying and/ortesting candidate therapeutics.

4.7.2. Cell Based and Cell Free Assays for Identifying Therapeutics

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements.

Accordingly, in an exemplary screening assay of the present invention,the compound of interest is contacted with a protein which may functionupstream (including both activators (enhancers) and repressors of itsactivity) or to proteins and/or nucleic acids (e.g promoter) which mayfunction downstream of the EFEMP1 polypeptide, whether they arepositively or negatively regulated by it. To the mixture of the compoundand the upstream or downstream element is then added a compositioncontaining an EFEMP1 polypeptide. Detection and quantification ofcomplexes of EFEMP1 with it's upstream or downstream elements provide ameans for determining a compound's efficacy at antagonizing (inhibiting)or agonizing (potentiating) complex formation between an EFEMP1 proteinand an EFEMP1 binding element (e.g. protein or nucleic acid). Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, isolated and purifiedEFEMP1 polypeptide is added to a composition containing the EFEMP1binding element, and the formation of a complex is quantitated in theabsence of the test compound.

Complex formation between the EFEMP1 polypeptide and a binding elementmay be detected by a variety of techniques. Modulation of the formationof complexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled EFEMP1 polypeptides, by immunoassay, or by chromatographicdetection.

Typically, it will be desirable to immobilize either EFEMP1 protein orits binding protein to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of EFEMP1 to an upstream ordownstream element, in the presence or absence of a candidate agent, canbe accomplished in any vessel suitable for containg the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/EFEMP1 (GST/EFEMP1) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates, e.g. an ³⁵S-labeled, and the testcompound, and the mixture incubated under conditions conducive tocomplex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintillant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofEFEMP1-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, an EFEMP1 proteinor its cognate binding protein can be immobilized utilizing conjugationof biotin and streptavidin. For instance, biotinylated molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with EFEMP1or with a protein encoded by a, gene that is up- or down-regulated byEFEMP1 can be derivatized to the wells of the plate, and protein trappedin the wells by antibody conjugation. As above, preparations of abinding protein and a test compound are incubated in the proteinpresenting wells of the plate, and the amount of complex trapped in thewell can be quantitated. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theprotein binding element, or which are reactive with the EFEMP1 protein;as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the binding element, either intrinsic orextrinsic activity. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with the bindingpartner. To illustrate, the binding partner can be chemicallycross-linked or genetically fused with horseradish peroxidase, and theamount of polypeptide trapped in the complex can be assessed with achromogenic substrate of the enzyme, e.g. 3,3′-diamino-benzadineterahydrochloride or 4-chloro-1-napthol. Likewise, a fusion proteincomprising the polypeptide and glutathione-S-transferase can beprovided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al. (1974) J BiolChem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein can beused. Alternatively, the protein to be detected in the complex can be“epitope tagged” in the form of a fusion protein which includes, inaddition to the EFEMP1 sequence, a second polypeptide for whichantibodies are readily available (e.g. from commercial sources). Forinstance, the GST fusion proteins described above can also be used forquantification of binding using antibodies against the GST moiety. Otheruseful epitope tags include myc-epitopes (e.g., see Ellison et al.(1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequencefrom c-myc, as well as the pFLAG system (International Biotechnologies,Inc.) or the pEZZ-protein A system (Phararnacia, N.J.). Transcriptionfactor-DNA binding assays are described in U.S. Pat. No. 5,563,036,which is owned by Tularik and is specifically incorporated by referenceherein.

Further, an in vitro assays can be used to detect compounds which can beused for treatment of diseases, such as MD, which are caused orcontributed to by defective or deficient EFEMP1 genes or proteins. Forexample, cells can be engineered to express an EFEMP1 gene (wildtype ormutant) in operative linkage with a reporter gene construct, such asluciferase or chloramphenicol acetyl transferase, or other reporter geneknown in the art. Cells can then be contacted with test compounds andthe rate or level of EFEMP1 expression can be assayed to identifyagonists or antagonists.

Also, a DNA footprinting assay can be used to detect compounds whichalter the binding of an EFEMP1 protein to nucleic acids (see forexample, Zhong et al. 1994 Mol. Cell Biol. 14:7276). Further, EFEMP1 maybe translationally or post-translationally modified by processes such asmRNA editing or protein truncation. Assays to specifically monitor theseprocesses can be performed according to protocols, which are well-knownin the art.

In addition to cell-free assays, such as described above, the EFEMP1proteins provided by the present invention also facilitates thegeneration of cell-based assays for identifying small moleculeagonists/antagonists and the like. For example, cells can be caused tooverexpress a recombinant EFEMP1 protein in the presence and absence ofa test agent of interest, with the assay scoring for modulation inEFEMP1 responses by the target cell mediated by the test agent. As withthe cell-free assays, agents which produce a statistically significantchange in EFEMP1-dependent responses (either inhibition or potentiation)can be identified.

Exemplary cell lines may include retinal pigment epithelial cell lines.Further, the transgenic animals discussed herein may be used to generatecell lines, containing one or more cell types involved in MD, that canbe used as cell culture models for this disorder. While primary culturesmay be utilized, the generation of continuous cell lines is preferred.For examples of techniques which may be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648.

For example, the effect of a test compound on a variety of end pointscould be tested. Similarly, epithelial cells can be treated with testcompounds or transfected with genetically engineered EFEMP1 genes.Monitoring the influence of compounds on cells may be applied not onlyin basic drug screening, but also in clinical trials. In such clinicaltrials, the expression of a panel of genes may be used as a “read out”of a particular drug's therapeutic effect.

In yet another aspect of the invention, the subject EFEMP1 polypeptidescan be used in a “two hybrid” assay (see, for example, U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), for isolating coding sequences for other cellular proteinswhich bind to or interact with an EFEMP1(e.g., EFEMP1 binding proteins”or “EFEMP1bp”).

Briefly, the two hybrid assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for an EFEMP1polypeptide. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a sample gene from a cDNA library.If the bait and sample hybrid proteins are able to interact, e.g., forman EFEMP1 dependent complex, they bring into close proximity the twodomains of the transcriptional activator. This proximity is sufficientto cause transcription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the reporter gene can be detected and usedto score for the interaction of the EFEMP1 and sample proteins.

4.7.3 Transgenic Animals For Identifying Therapeutics

Transgenic animals can also be made to identify therapeutics thatmodulate an EFEMP1 bioactivity, to confirm the safety and efficacy of acandidate therapeutic or to study drusen formation. Transgenic animalsof the invention can include non-human animals containing a mutatedEFEMP1 gene under the control of an appropriate homologous orheterologous promoter.

Methods for obtaining transgenic non-human animals are well known in theart. In preferred embodiments, the expression of the mutation isrestricted to specific subsets of cells, tissues or developmental stagesutilizing, for example, cis-acting sequences that control expression inthe desired pattern. In the present invention, such mosaic expressioncan be essential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, expression levelwhich might grossly alter development in small patches of tissue withinan otherwise normal embryo. Toward this end, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the mutation in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences. Genetic techniques, which allow for the expressionof the mutation can be regulated via site-specific genetic manipulationin vivo, are known to those skilled in the art.

The transgenic animals of the present invention all include within aplurality of their cells a mutant EFEMP1 transgene of the presentinvention, which transgene alters the phenotype of the “host cell”. Inan illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation of expression of a mutation containing transgene can beregulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of amutation containing transgene requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and themutation transgene can be provided through the construction of “double”transgenic animals. A convenient method for providing such animals is tomate two transgenic animals each containing a transgene.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the transactivatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, the transgene could remain silent into adulthooduntil “turned on” by the introduction of the transactivator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal Embryonal target cells at various developmentalstages can be-used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-29g haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed). In one embodiment, thetransgene construct is introduced into a single stage embryo. The zygoteis the best target for microinjection. In the mouse, the male pronucleusreaches the size of approximately 20 micrometers in diameter whichallows reproducible injection of 1-2 pl of DNA solution. The use ofzygotes as a target for gene transfer bas a major advantage in that inmost cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus. For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous, genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material. Further, in such embodimentsthe sequence will be attached to a transcriptional control element,e.g., a promoter, which preferably allows the expression of thetransgene product in a specific type of cell.

Retroviral infection can also be used to introduce the transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jalner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

4.8 Methods of Treatment

4.8.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds identified asdescribed above can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining The LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissues inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

4.8.2. Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For parenteral administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable delivery, systems includemicrospheres which offer the possibility of local noninvasive deliveryof drugs over an extended period of time. This technology utilizesmicrospheres of precapillary size which can be injected via a coronarycatheter into any selected part of the e.g. heart or other organswithout causing inflammation or ischemia. The administered therapeuticis slowly released from these microspheres and taken up by surroundingtissue cells (e.g. endothelial cells).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The compositions may, if desired, be presented in a pack, or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application) arehereby expressly incorporated by reference.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques that are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, (2nd ed., Sambrook, Fritsch andManiatis, eds., Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984)

EXAMPLE 1

Methods and Materials

DNA isolation. A total of 1163 individuals were studied: 166 individualsaffected with either ML or DHRD, 26 unaffected spouses of the ML or DHRDfamilies, 494 AMD patients, and 477 control patients. Of the 494 AMDpatients, 318 were ascertained in Iowa, 87 elsewhere in the US, 25 inAustralia, and 64 in Switzerland. The control samples were obtained fromunrelated individuals who were not known to have macular degeneration.The purpose of these samples was to allow an estimation of the generalpopulation frequency of any sequence changes observed in the ML/DHRD andAMD groups. Of the control individuals, 104 were ascertained in Iowa,100 in Australia, 187 in Switzerland, and 86 elsewhere in the US. DNAwas extracted from venous blood using a previously described protocol⁷⁰.YAC, BAC, and plasmid DNA was isolated using a previously describedprotocol⁷¹.

Marker typing. Short Tandem Repeat Polymorphisms (STRPs) were analyzedwith PCR amplification and gel electrophoresis as previously described⁷¹. Six of these STRPs as well as four intragenic EFEMP1 polymorphismshave not been previously reported (see Table 1). Followingelectrophoresis, the gels were stained with silver nitrate. STS contentanalysis was performed as previously described⁷¹ and used it to deducethe minimum tiling path of the 2 p Malattia Leventinese interval.

YAC and BAC identification and subcloning. YACs were initiallyidentified from the critical region by searching a database at theWhitehead Institute/MIT Genome Center with STSs known to lie in theinterval. Additional YACS and BACs were then identified with PCR-basedscreening of pooled libraries (Research Genetics) with various STSs fromthe interval. BAC DNA was subcloned as previously described⁷¹.

Sequencing of plasmids and PCR products. PCR was performed forsequencing in a 30 μl reaction and the products were purified with aConcert Rapid PCR purification kit (Gibco-BRL). 500 ng of plasmid DNA or3.0 μl of PCR product was used as template for a sequencing reaction aspreviously described⁷¹.

Gene characterization. The genomic organization of EFEMP1 was determinedwith long range and vectorette PCR using the Expand™ Long Template PCRsystem (Boehringer Mannheim) and a vectorette cassette (Genosys). ThePCR products were sized on 1-2% agarose gels with a 100 bp ladder(Gibco/BRL). These PCR products were sequenced and the result comparedto published cDNA and exon-intron junction sequence^(62,72) to confirmexon-intron borders. This new intronic sequence was used to designprimers for SSCP and DNA sequencing of the coding regions using thePrimer 3 program(http://www-genome.wi.mit.edu/genome_Software/other/primer3.html).

RNA isolation, blot analysis and RT-PCR. A mouse poly (A) mRNA Northernblot was prepared with RNA from freshly dissected adult tissues. Thisblot was hybridized using a previously described protocol⁷³ with a 920bp gel purified insert of the murine EFEMP1 cDNA plasmid (I.M.A.G.E.Consortium Clone ID 1480170, Research Genetics) corresponding to the 3′region of the gene. Human donor eyes were obtained from the Iowa LionsEye Bank (Iowa City, Iowa) within 4 hours of death. Six-mm trephinepunches of RPE were removed and total RNA was isolated using the RNeasykit (Qiagen). cDNA was synthesized from this RNA with random primers andthe cDNA was used in PCR analyses. After 20-35 cycles of PCR, thesereaction mixtures were analyzed with agarose gel electrophoresis.

Mutation detection and confirmation. Mutation screening was performedusing single strand polymorphism (SSCP) analysis and direct sequencingof PCR products as previously described.⁷¹ The primer sequences used forthis screening are given in Table I.

Results and Discussion

Previously, 5 families with the ML phenotype with a total of 56 affectedmembers were investigated. To narrow the genetic interval containing theML/DHRD gene 28 additional families (75 patients) affected with eitherML or DHRD were ascertained from the United States and Switzerland.These families were genotyped with 19 STRP markers in the diseaseinterval but did not observe any recombination events that would narrowthe disease interval more than that reported by Gregory, et al.⁴⁸ (FIG.2). However, haplotypic analysis of 23 Swiss families that were likely(for geographic reasons) to share a common ancestor did reveal anarrower interval: between D2S2352 and D2S378 (FIG. 2). Five Australianfamilies were later studied, including one with a clearly affectedmember who was recombinant telomeric to 293JI2CA, which further narrowedthe interval to the segment between 293J12CA and D2S378 (FIG. 2).Markers from this interval were then used to screen libraries of yeastand bacterial artificial chromosomes, and contigs were assembled (FIG.2) for verification of the genetic map as well as for evaluation ofcandidate genes and expressed sequence tags (ESTs). In an attempt toidentify additional informative polymorphisms, twelve ESTs were screenedand six STSs for polymorphisms in a single amplimer in ML, DHRD and AMDpatients. In addition, six genes were screened (EFEMP1, beta-fodrin,ubiquitin, WI-6613, WI-22280, and WI-1332) for coding sequencemutations. Only one novel sequence variation was identified during thisphase of the project: a single base pair change in the 3′ UTR of WI-6613that was present in all Swiss families but absent from American MLfamilies.

EFEMP1, beta-fodrin, and WI-6613 were then screened more comprehensivelyfor mutations, since they were each present in retinal cDNA librariesand mapped within the narrowest genetic interval. The genomic structuresand intronic sequences of these genes were then determined and theircoding regions evaluated for mutations by both SSCP analysis and directDNA sequencing. Beta-fodrin and WI-6613 were each approximately 70%screened without detecting any amino-acid-altering sequence variationswhen a potential disease-causing variation in EFEMP1 was detected. ThisC to T transition (Arg345Trp-FIG. 3) was initially observed in 5families with ML (2 from the US, 2 from Switzerland, and 1 fromAustralia). The potential involvement of this variation in ML, DHRD, andAMD was then assessed by SSCP screening of all 162 affected patients inthe 37 families available at that time, as well as 477 controlindividuals and 494 unrelated patients affected with AMD. Of the 162patients initially thought to be affected with ML or DHRD, 161 werefound to harbor an SSCP shift in exon 10 of EFEMP1. None of the AMD orcontrol individuals exhibited this shift. Sequence analysis of onepatient exhibiting an SSCP shift from each of the 37 families revealedall to harbor the Arg345Trp mutation. The retinal photographs of the two“affected” members of the single family that was discordant for theArg345Trp change was then reexamined and it was discovered that theindividual with the mutation had the characteristic ML phenotype (andthe shared Swiss haplotype) while the individual lacking the mutationhad a phenotype more typical of common AMD (and failed to share alleleswith the Swiss haplotype). Of the 161 ML/DHRD patients harboring theArg345Trp mutation, 160 carried it in the heterozygous state. Oneindividual was homozygous for this change (FIG. 3) and had a retinalphenotype that was neither more nor less severe than heterozygotes ofsimilar age.

STS content analysis from additional BACs allowed a revision of themarker order such that all 25 Swiss families shared a four markerhaplotype that included the Arg345Trp variant. This “final Swissinterval” (FIG. 2) completely excluded beta-fodrin and WI-6613, leavingEFEMP1 as the sole occupant of the critical interval among the group of12 ESTs and six genes originally considered as candidates.

The entire EFEMP1 coding sequence was then screened by SSCP in 494 AMDpatients and 477 control individuals. Three additional EFEMP1 codingsequence variations were detected (Thrl81 Thr, ACG-ACA; Ile220Phe,ATC-TTC; and Ser456Ser, TCA-TCG), each present in a single controlindividual. The entire coding sequence of EFEMP1 was then sequenced inboth directions in four individuals: 2 probands with ML (one fromSwitzerland and one from the United States), one proband with DHRD (fromAustralia), and a general population control. No additional codingsequence variations were detected.

To investigate the possibility that the original DHRD family harbored adifferent mutation in the EFEMP1 gene, samples from two nuclear families(one from Northern Ireland and one from England) with genealogicalevidence for a relationship with Doyne's original family were studied.⁶⁰Affected individuals from both of these families were found to harborthe Arg345Trp variation.

In an attempt to identify haplotypic recombinants within EFEMP1, a 7.5kb of intronic sequence was screened for polymorphisms with SSCP. Fourintragenic two-allele polymorphisms were identified (see methods) andall 39 families were found to carry the most common allele of each inphase with the Arg345Trp variant.

Although retinal expression of EFEMP1 can be inferred from its presencein retinal cDNA libraries, FIG. 4 a, a northern blot of RNA from tissuesfrom adult mice, revealed EFEMP1 to be abundantly expressed in the eyeand the lung, and moderately expressed in the brain, heart, spleen, andkidney. An RT-PCR on RNA extracted from isolated retinal pigmentepithelial (RPE) cells, a preparation of RPE and choroid, and isolatedneurosensory retina (all obtained from human donors) revealed EFEMP1 tobe expressed in all of these tissues (FIG. 4 b).

The absence of de novo Arg345Trp mutations in the 39 families studied,and the complete sharing of alleles of four intragenic EFEMP1polymorphisms among these families suggest that the Arg345Trp mutationoccurred only once, in a common ancestor of every affected patient inthis study. Despite the extensive patient resources available, we wereunable to demonstrate that the ancestrally shared interval is entirelycontained within the EFEMP1 gene and thus the possibility of adisease-causing mutation in an adjacent gene can not be completelyexcluded. However, the striking linkage disequilibrium between thedisease phenotype and the Arg345Trp variant (absent from over 1900alleles of non-ML/DHRD individuals) and the high degree of conservationof the EFEMP1 gene (Arg345Trp is the only nonconservative amino acidchanging variant observed in the 1163 individuals in this study andalters a codon that is conserved among human, mouse and rat) providestrong evidence that Arg345Trp is disease-causing. Moreover, EFEMP1 is aplausible candidate gene. It is expressed in the tissues closest to thesite of drusen formation (RPE and retina) and encodes a protein that ishomologous to a family of extracellularmatix glycoproteins known asfibulins⁶¹ . EFEMP1 was originally isolated as a cDNA sequence (thenknown as S1-5) that was relatively over-expressed in human fibroblastsobtained from a patient with Werner syndrome, a genetic diseasecharacterized by accelerated aging⁶². Finally, the Arg345Trp mutationalters the last EGF domain of the EFEMP1 gene product. It is similar toa number of fibrillin mutations that cause Marfan syndrome^(63,64).TABLE 1 Primer sequences EFEMPI gene assay FORWARD REVERSE EXON 3 5′GTTTTGTTACTTTCCCCGCA 3′ 5′ ACTGGCAGGGGTGTGTAAAG 3′ (SEQ ID NO:3) (SEQ IDNO:4) EXON 4 5′ CCAATTAACTGTCTCCTGGC 3′ 5′ AAGGCAATGATCACATGGAAG 3′ (SEQID NO:5) (SEQ ID NO:6) EXON 5A 5′ CATGTTTGATTTTCCCTCTTAGAA 3′ 5′ATGCTGCTGGCAGCTACAACC 3′ (SEQ ID NO:7) (SEQ ID NO:8) EXON 5B 5′AACCTCAGGGGCAACCAC 3′ 5′ TTCAATGGTTAGGAAAAGAAGTTATTC 3′ (SEQ ID NO:9)(SEQ ID NO:10) EXON 6 5′ TGACAATTCTTTCTGTGTTGCAT 3′ 5′CTCAAGACAGGACCGTGGTC 3′ (SEQ ID NO: 11) (SEQ ID NO: 12) EXON 7 5′TTCTCTTTGTGTGTGTGCCTG 3′ 5′ TGGGGTTTCCTTTTGTGAAG 3′ (SEQ ID NO:13) (SEQID NO:14) EXON 8 5′ CAAAAGAGTAAGGATATGTTTAAAGTC 3′ 5′GGACTTTATTCCATACTATCTGGG 3′ (SEQ ID NO:15) (SEQ ID NO:16) EXON 9 5′TGGTGCACAAACTTTTCAACTC 3′ 5′ TCCTCTTGTCTCTTCCTGGC 3′ (SEQ ID NO:17) (SEQID NO: 18) EXON 10 5′ CTTGCAAACAGAATCTGCCA 3′ 5′TCCTCACTTTCAAAAGTTCTGATTT 3′ (SEQ ID NO:19) (SEQ ID NO:20) EXON 11A 5′ACCAAGCCAAACTGCTGAAT 3′ 5′ AAAAGTATTGATGGTGTTGGCA 3′ (SEQ ID NO:21) (SEQID NO:22) EXON 11B 5′ TGCCATCAGACATCTTCCAG 3′ 5′ AATGTTTGCTTTCCTTCCACA3′ (SEQ ID NO:23) (SEQ ID NO:24) EXON 12 5′ GCATAGAAACTCCAATCCAAGAA 3′5′ TGCCTGTGGTTGACTCTTAGAA 3′ (SEQ ID NO:25) (SEQ ID NO:26) Novel repeats293J12CA 5′ GGAACAAGCAGGACCTTTCA 3′ 5′ TGTTATATCCTATTTGAGCT 3′ (SEQ IDNO:27) (SEQ ID NO:28) 322A4AAAT 5′ ATCCTAGCAAAACATAAGAGT 3′ 5′CTTACATTCCTGTGGACTTGA 3′ (SEQ ID NO:29) (SEQ ID NO:30) 133018CA 5′CGGGGATCTTTTTCATGATG3′ 5′ GGGGCAAGGCAAGAGTAAG3′ (SEQ ID NO:31) (SEQ IDNO:32) 133018AAAT 5′ CTGCAGTGAGCTGCGATTAT3′ 5′ TTTTGCTTTGGGAATTAGCAG3′(SEQ ID NO:33) (SEQ ID NO:34) 340018CA 5′ GGAGGTTGCAGTGAGCTG 3′ 5′TTGAATTGTCGTGAATCTTGTT3′ (SEQ ID NO:35) (SEQ ID NO:36) 202J12GGAA[5′TACCACTGCACTGAAGCCTG3′ 5′AAATCTTCTGCAAAAACAAAAGTG3′ (SEQ ID NO:37)(SEQ ID NO:38) Intragenic polymorphisms Intron 4 5′ CCAATTAACTGTCTCCTGGC3′ 5′ TTTGTGCACCACTACTTTGGA 3′ (SEQ ID NO:39) (SEQ ID NO:40) Intron 8 5′AAATGTGCCCAAGTCACACA 3′ 5′ TTTGAAACTGGACCCAAGG3′ (SEQ ID NO:41) (SEQ IDNO:42) Intron 9 5′ AGCATAAGCTCAATATGGGAGT3′ 5′ TGGCAGTGTTACCAAGAGGA 3′(SEQ ID NO:43) (SEQ ID NO:44) Intron 11 5′CAACACCATCAATACTTTTCGG3′ 5′AAGGCAATGATCACATGGAAG 3′ (SEQ ID NO:45) (SEQ ID NO:46)References

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1. A method for identifying a compound that modulates a EFEMP1bioactivity, comprising the steps of: (a) contacting an appropriateamount of the compound with a cell or cellular extract, which expressesan EFEMP1 gene; and (b) determining the resulting EFEMP1 bioactivity,wherein an increase or decrease in the EFEMP1 bioactivity in thepresence of the compound as compared to the bioactivity in the absenceof the compound indicates that the compound is a modulator of an EFEMP1bioactivity.
 2. A method of claim 1, wherein the EFEMP1 gene is a humanEFEMP1 gene.
 3. A method of claim 1, wherein the EFEMP1 gene is awildtype gene.
 4. A method of claim 1, wherein the EFEMP1 gene is amutant gene.
 5. A method of claim 1, wherein the modulator is an agonistof an EFEMP1 bioactivity.
 6. A method of claim 1, wherein the modulatoris an antagonist of an EFEMP1 bioactivity.
 7. A method of claim 1,wherein in step (b), the EFEMP1 bioactivity is determined by determiningthe expression level of an EFEMP1 gene.
 8. A method of claim 7, whereinthe expression level is determined by detecting the amount of mRNAtranscribed from an EFEMP1 gene.
 9. A method of claim 7, wherein theexpression level is determined by detecting the amount of EFEMP1 geneproduct produced.
 10. A method of claim 9, wherein the expression levelis determined using an anti-EFEMP1 antibody in an immunodetection assay.11. A method of claim 1, which additionally comprises the step ofpreparing a pharmaceutical composition from the compound.
 12. A methodof claim 1, wherein said cell is contained in an animal.
 13. A method ofclaim 12, wherein the animal is transgenic.
 14. A method of claim 13,wherein the transgenic animal contains a human EFEMP1 gene.
 15. Acompound identified by the method of claim
 1. 16. A compound of claim15, which is selected from the group consisting of: a small molecule, apolypeptide, a nucleic acid and a peptidomimetic.
 17. A compound ofclaim 16, wherein the nucleic acid is selected from the group consistingof: an antisense molecule, a ribozyme and a triplex nucleic acid.
 18. Acompound of claim 16, wherein the polypeptide is a EFEMP1 polypeptide.19. A method for identifying whether a test molecule is an EFEMP1binding partner or measuring the strength of an interaction between anEFEMP1 polypeptide and said EFEMP1 binding partner comprising: (a)allowing (i) a first molecule comprising a EFEMP1 polypeptide operablylinked to a heterologous DNA binding domain to interact with (ii) asecond molecule comprising a test molecule operably linked to apolypeptide transcriptional activation domain and (iii) a hybridreporter gene comprising a nucleic acid encoding a reporter operablylinked to a DNA sequence comprising a binding site for said heterologousDNA binding domain; and (b) detecting or measuring the expression of thehybrid reporter gene as an indication of the existence or strength of aninteraction between the first molecule and the second molecule whereinhigh levels of hybrid reporter expression indicate a strong interactionbetween EFEMP1 and said test molecule thereby identifying a testmolecule which is an EFEMP1 binding partner.
 20. A method of claim 19,wherein said second molecule is encoded by a nucleic acid and comprisesa test polypeptide operably linked to a polypeptide transcriptionalactivation domain, and which further comprises the step of isolating thenucleic acid encoding said second molecule from a cell expressing thehybrid reporter gene.
 21. A method for identifying a molecule which is adownstream or an upstream component of an EFEMP1 biochemical pathway orfor measuring the strength of the interaction between a EFEMP1biochemical pathway component and a EFEMP1 binding partner comprising:(a) allowing (i) a first molecule comprising a EFEMP1 binding partnerpolypeptide operably linked to a heterologous DNA binding domain tointeract with (ii) a second molecule comprising a test molecule operablylinked to a polypeptide transcriptional activation domain and (iii) ahybrid reporter gene comprising a nucleic acid encoding a reporteroperably linked to a DNA sequence comprising a binding site for saidheterologous DNA binding domain; and (b) detecting or measuring theexpression of the hybrid reporter gene as an indication of the existenceor strength of an interaction between the first molecule and the secondmolecule wherein high levels of hybrid reporter expression indicate astrong interaction between a EFEMP1 binding partner and said testmolecule thereby identifying a test molecule which is a downstream or anupstream component of the EFEMP1 biochemical pathway.
 22. A method ofclaim 21, wherein said second molecule is encoded by a nucleic acid andcomprises a test polypeptide operably linked to a polypeptidetranscriptional activation domain, and which further comprises the stepof isolating the nucleic acid encoding said second molecule from a cellexpressing the hybrid reporter gene.
 23. A method for identifying acompound, which interacts with a EFEMP1 polypeptide or EFEMP1 bindingpartner, comprising the steps of: (a) contacting an appropriate amountof the compound with a EFEMP1 polypeptide and a EFEMP1 binding partnerunder conditions wherein, but for the test compound, the EFEMP1polypeptide and EFEMP1 binding partner are able to interact; and (b)detecting the extent to which a EFEMP1 polypeptide/EFEMP1 bindingpartner complex is formed in the presence of the compound, wherein anincrease or decrease in the amount of complex formed in the presence ofthe compound relative to in the absence of the compound indicates thatthe compound interacts with a EFEMP1 polypeptide or EFEMP1 bindingpartner.
 24. A method of claim 23, wherein the EFEMP1 polypeptide is ahuman EFEMP1 polypeptide.
 25. A method of claim 23, wherein the EFEMP1polypeptide is a wildtype polypeptide.
 26. A method of claim 23, whereinthe EFEMP1 polypeptide is a mutant polypeptide.
 27. A method of claim23, wherein the compound, which interacts with a EFEMP1 polypeptide orEFEMP1 binding partner is a EFEMP1 agonist.
 28. A method of claim 23,wherein the compound, which interacts with a EFEMP1 polypeptide orEFEMP1 binding partner is a EFEMP1 antagonist.
 29. A method of claim 23,which additionally comprises the step of preparing a pharmaceuticalcomposition from the compound.
 30. A compound identified by the methodof claim
 29. 31. A compound of claim 30, which is selected from thegroup consisting of: a small molecule, a polypeptide, a nucleic acid anda peptidomimetic.
 32. An isolated EFEMP1 nucleic acid which is operablylinked to a EFEMP1 transcriptional regulatory sequence.
 33. A nucleicacid of claim 32, wherein the EFEMP1 transcriptional regulatory sequenceis selected from the group consisting of: a EFEMP1 enhancer, a EFEMP1promoter, and a EFEMP1 initiator element.
 34. An isolated nucleic acidof claim 32, wherein the EFEMP1 nucleic acid is functionally fused to aheterologous gene.
 35. An isolated nucleic acid of claim 34, whereinsaid heterologous gene encodes a protein selected from the groupconsisting of a positive selectable marker, a negative selectable markerand a reporter gene.
 36. An isolated nucleic acid of claim 35, whereinthe coding sequence of EFEMP1 is disrupted by a positive selectablemarker.
 37. An isolated nucleic acid of claim 36, wherein said nucleicacid is further flanked by a negative selectable marker or markers. 38.An isolated nucleic acid of claim 37, wherein the reporter gene isselected from the group consisting of: beta-galactosidase andluciferase.
 39. A cell line comprising an isolated nucleic acid of claim39.
 40. An animal comprising an isolated nucleic acid of claim
 39. 41.An animal of claim 40, which is transgenic.
 42. An animal of claim 41,which contains a human EFEMP1 gene.
 43. An isolated nucleic acidcomprising a EFEMP1 responsive regulatory sequence operably linked to areporter gene.
 44. An isolated nucleic acid of claim 43, wherein thereporter gene is selected from the group consisting ofbeta-galactosidase and luciferase.
 45. A cell line comprising anisolated nucleic acid of claim
 43. 46. An animal comprising an isolatednucleic acid of claim
 43. 47. An animal of claim 46, which contains ahuman EFEMP1 gene.
 48. A cell in which the biological activity of one ormore EFEMP1 proteins is altered by a chromosomally incorporatedtransgene.
 49. A cell of claim 48, wherein said transgene disrupts atleast a portion of a genomic EFEMP1 gene.
 50. A cell of claim 48,wherein said transgene deletes all or a portion of the genomic EFEMP1gene by replacement recombination.
 51. A cell of claim 48, wherein saidtransgene comprises: (i) at least a portion of the genomic EFEMP1 gene,and (ii) a marker sequence which provides a detectable signal foridentifying the presence of the transgene in a cell.
 52. A transgenicanimal comprised of a cell in claim 48.